Rabies is a disease which affects humans and other mammals, including dogs and cattle.
Rabies is caused by a neurotrophic arbovirus of the rhabdovirus family, Lyssavirus genus.
The virus is transmitted from victim to victim in saliva. Once inoculated into a new host via a bite by a rabid animal, the virus concentrates at neuromuscular junctions, where it is taken up into nerve cells. Watson et al., J. Gen. Virol. 56, 371(1981); Lentz et al., Science 215, 182-184(1982). Once inside a nerve cell, the virus is transported in the retrograde direction along nerve processes and eventually reaches the spinal cord and brain. The virus replicates in and is released from spinal cord and brain cells. Upon release from nerve cells, the virus travels to the salivary glands, where it infects the acinar cells, from which virus is released into the saliva in very high numbers. The virus, at high titer in the saliva, can then be inoculated by a bite into another animal.
Most importantly, replication of the virus in the brain cells destroys the cells and produces an encephalitis, which may eventually kill the victim.
Blocking access of rabies virus to nerve cells of a mammal bitten by a rabid animal would block development of the disease in the former.
A person exposed to rabies virus, typically through an animal bite, is treated today in three ways. First, the bite wound is thoroughly washed with soap and water. Second, as soon as possible after the exposure, human antirabies gamma globulin, isolated from plasma of human donors hyperimmunized with inactivated rabies virus vaccine, is administered by intramuscular injection and is thoroughly infiltrated into tissue at and around the wound site. If human gamma globulin is not available, equine antirabies serum is used instead. The gamma globulin or serum formulation provides a passive anti-rabies immunity, because it contains antibodies which bind to rabies virus and facilitate virus removal from the bitten individual's system before the virus can establish itself by invasion of the person's nerve cells. The passive immunity conferred by the anti-rabies gamma globulin of anti-serum has a half-life of about 3 weeks. Third, in addition to the other steps, the person is vaccinated by intramuscular injection of five or six doses of inactivated rabies virus, the first vaccination being as soon as possible after the exposure and the others at 3, 7, 14, 28 and, optionally, 90 days after the first. Beginning 7 to 10 days after the first dose, the vaccine causes the person to mount an active immune response against the rabies virus; antibodies produced in this response mediate neutralization of the virus before it can become established in the exposed individual. See ACIP (Immunization Practices Advisory Committee) Recommendations on Rabies Prevention, Morbidity and Mortality Weekly Report 33, 393 (July 20, 1984) (Centers for Disease Control, Atlanta, Ga., U.S.A.).
As a prophylactic measure, persons subject to high risk of infection with rabies virus, via contact with rabid animals, work with rabies virus or otherwise, may be immunized against the virus with inactivated-virus vaccines. See ACIP (July 20, 1984), supra.
As a means of rabies control, domesticated mammals, such as dogs, cats, horses, and cattle, may be immunized against rabies virus by vaccination by injection with inactivated or attenuated virus or with the coat glycoprotein isolated from the virus. Oral vaccines, based on attenuated virus, have been developed and used in attempts to immunize populations of wild animals, such as foxes, against rabies.
There are a number of risks associated with the currently available, anti-rabies vaccines. The vaccines which contain inactivated or attenuated virus occasionally produce neurologic or central nervous system disorders in those vaccinated. Further, there is a risk that all of the virus in a lot of supposedly inactivated-virus vaccine will not be killed, or that some of the virus in a lot of attenuated-virus vaccine will revert to a virulent state, and that rabies might be caused in an individual mammal by vaccination with a dose which happens to contain live, virulent virus. A sythetic-peptide based anti-rabies vaccine would not involve these risks and would be substantially safer than the currently available vaccines.
Vaccines based on coat glycoprotein isolated from the virus entail many of the risks associated with inactivated- or attentuated-virus vaccines, because obtaining coat glycoprotein involves working with live virus.
Antisera, from which anti-rabies gamma globulin is derived for use in conferring passive immunity against the disease on persons who have been exposed to live virus, is taken from humans who have been hyperimmunized with inactivated rabies virus, as indicated above. Thus, obtaining anti-rabies gamma globulin is at least as risky as vaccination with currently available vaccines.
Obtaining antisera requires withdrawing blood, which also entails some risk. Further, administration of fractions from antisera entails a number of dangers similar to those associated with blood transfusion.
The risks associated with obtaining and using anti-rabies gamma globulin could be avoided by producing antibodies, effective to block establishment of rabies in individuals exposed to live rabies virus, in large quantity in substantially pure form, as by culturing hybridomas which secrete such antibodies. These risks would be reduced even further if production of the hybridomas was based on immunization of mammals, or in vitro immunization of mammalian lymphocytes, with synthetic, peptide-based, anti-rabies vaccines, which, as indicated supra, entail far less risk than vaccines based on inactivated or attenuated virus or proteins obtained from cultured virus.
Synthetic vaccines are known wherein an haptenic, synthetic peptide is linked to an immunogenic carrier molecule, which induces a mammal's immune system to produce antibodies against the haptenic peptide. The haptenic peptide in such vaccines has an amino acid sequence which includes a sequence which is the same as, or closely similar to, the sequence of an antigenic determinant on the toxin or infectious agent against which the vaccine is intended to provide protection. It has been found that some of the antibodies, raised in a mammal immunized with such a synthetic vaccine, will bind to the corresponding antigenic determinant on the surface of the toxin or infectious agent. Thus, such vaccines provide immune protection against intoxication or infection with toxin or infectious agent against which the vaccine is directed. The key to developing such a vaccine is identifying an antigenic determinant, on the toxin or infectious agent, which has a sequence of amino acids that is continuous, i.e., the determinant is an uninterrupted fragment of the primary structure of the protein on which the determinant occurs.
Typically, numerous strains of a virus will occur naturally. The various strains may be antigenically variable, i.e. differ from one another in the amino acid sequences of one or more of their antigenic determinants. Thus, a vaccine based on a single strain of a virus may not provide immunity in a vaccinated individual against other strains of the same virus, as antibodies induced by the single strain may not be reactive with antigenic determinants on other strains. Despite having been vaccinated, such an individual will remain susceptible to the disease caused by the virus. This problem of antigenic variability has in fact been encountered with currently available anti-rabies vaccines. Thus, in a synthetic vaccine, it is advantageous to use as the synthetic peptide one which includes an amino acid sequence identical, or closely similar, to that of an antigenic determinant on the target virus which cannot differ in sequence among strains of the virus. Such determinants will be those which are essential to propagation of the virus and which cannot fulfill their essential role in propagation if their sequences change. The sequences of such determinants will be highly conserved among all strains of a virus. Thus, a vaccine with a synthetic peptide with such a sequence will not be limited by antigenic variability and will be effective to provide protection against all strains of the virus against which the vaccine is intended to provide protection. Of course, making such a vaccine requires identifying an antigenic determinant on the virus which has a sequence that is highly conserved among the various strains.
Lentz et al., supra, have shown that the rabies virus particle attaches to muscle cells in culture and that the distribution of virus parallels the distribution of acetylcholine receptor. Further, Lentz et al. have reported data which indicate that rabies virus accumulates at the neuromuscular junction by binding to the acetylcholine receptors (AChR's) at such junction. Among the findings reported by Lentz et al. is that binding of rabies virus at the neuromuscular junction can be blocked by pre-incubation of tissue including such junctions with the curaremimetic, snake-venom neurotoxin, alpha-bungarotoxin, which is known to bind tightly to the acetylcholine (ACh) binding site of the AChR.
The rabies virus particle is bullet-shaped. Its negative-strand RNA genome is enveloped by an outer protein covering from which numerous "spikes" protrude which are molecules of the rabies virus coat glycoprotein (G protein). This glycoprotein is responsible for the immunogenicity of the virus. Thus, antigenic determinants of the virus are on the G protein.
Monoclonal antibodies have been prepared using various strains of whole rabies virus. Flamand et al., J. Gen. Virol.48, 105(1980).
Antibodies raised against the coat glycoprotein can neutralize infectious virus. See Dietzschold et al., Dev. Biol. Stand. 40, 45(1978).
The sequences of the coat glycoproteins from two strains of rabies virus are known. Anilionis et al., Nature 294, 275-278(1981); Yelverton et al., Science 219, 614-620(1983). These glycoproteins, in mature form, as they occur in the coat, of the virus outside infected cells, have sequences of 505 amino acids. The sequences are approximately 90% homologous.
Numerous curaremimetic, snake-venom neurotoxins, similar to alpha-bungarotoxin, are known which bind with high affinity at the ACh binding site of the AChR at neuromuscular junctions. The sequences of more than 60 of these neurotoxins are known. Studies of these neurotoxins, involving three-dimensional structural determinations, chemical modifications, and comparisons of sequences, have led to identification of four highly conserved amino acids which interact to form and stabilize a structure, which is similar to that of ACh and is thought to be involved in the binding of the neurotoxins to the active-site (i.e., the ACh binding-site) of the AChR. According to the amino acid numbering system based on the alignment of neurotoxin sequences by Karlsson (Handbook of Experimental Pharmacology 52, 159-212(1979), these four residues are tryptophan at position 29, aspartate at position 31, arginine at position 37 and glycine at position 38. The tryptophan at position 29 is thought to stabilize an ion-pair formed between the carboxylate group of aspartate-31 and the guanidinium group of arginine-37. It is this ion-pair which is thought to stereochemically mimic acetylcholine (Tsernoglou et al., Mol. Pharmacol. 14, 710(1978)). Modification of the tryptophan-29 results in a loss of toxic activity of no more than about 50% (Ryden et al., Int'l. Jour. Peptide Protein Res. 5, 261-273(1973)).